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My research interest is focused primarily on the biochemistry and molecular biology aspects of two tree-legumes, Leucaena leucocephala and Acacia koa. 

Leucaena leucocephala (leucaena):  Leucaena is a tree legume that grows easily in tropical and subtropical environments.  Its foliage is rich in protein; however, it contains a toxic non-protein amino acid, mimosine, which causes adverse side effects if the animals are fed with leucaena. Previously, we have isolated and characterized genes for mimosine degradation from Rhizobium TAL1145 that forms nitrogen-fixing root nodulates on leucaena.  Three important genes that we identified and characterized are midD, pydA and pydB, which encode rhizomimosinase, dioxygenase and hydrolase, respectively. We have also isolated a mimosine-degrading enzyme from leucaena. This enzyme, mimosinase, has similar catalytic properties as rhizomimosinase but the two enzymes have no sequence similarity with each other. We are currently working on site-directed mutational analysis of these two enzymes. We are collaborating with Dr. Yoshimitsu Kakuta’s laboratory at Kyusyu University on three-dimensional structure analysis of these two enzymes through X’-ray crystallography.

Acacia koa (koa): Acacia koa is an important leguminous tree endemic to the Hawaiian Islands. The native A. koa forests are broadly distributed across all five major Hawaiian Islands.  A few years ago, we discovered ‘canopy nodulation’, which is a new phenomenon of developing nitrogen-fixing nodules on the adventitious roots from the areal parts of the tree.  We also developed microsatellite markers for koa and applied these markers to determine genetic diversity in the koa populations of different Hawaiian Islands.  Then we developed a method of screening koa for resistance to wilt caused by Fusarium oxysporum. We have also cloned four classes of chitinase genes and used the gene sequences as a tool for molecular screening for resistance.  We are currently doing transcriptome analysis of koa.  Our focus is isolation of genes for resistance to biotic and abiotic stresses. 

Current graduate students:

1. Ahmed Bageel (2015- ) Ph.D. Research title: Characterization of genes for drought resistance in Leucaena leucocephala.
2. Michael Honda (2012- ) Ph.D. Research title: Expression of mimosinase in Leucaena leucocephala.
3. Maia Corpus (2015- ) M.S. Research title: Characterization of genes for tannin biosynthesis in Acacia koa.
4. James Carrillo (2015- ) M.S. Research title: Biochemical characterization of two mimosine-degradation pathway enzymes from Rhizobium. 
5. Christopher Nakano (2015- ) M.S. Research title: Expression of nicotianamine synthase in leucaena leucocephala.

Courses:

Research Interests :

Legume-Rhizobium symbiosis, tree legumes, mimosine metabolism

Publications :

Ishihara K, Lee EKW and Borthakur D (2017) Thigmomorphogenesis: changes in morphology, biochemistry, and levels of transcription in response to mechanical stress in Acacia koa. Can. J. For. Res. 47: 583–593 dx.doi.org/10.1139/cjfr-2016-0356

Negi, VS, Borthakur D (2016) Heterologous expression and characterization of mimosinase from Leucaena leucocephala. Methods in Molecular Biology 1405:59-77. doi: 10.1007/978-1-4939-3393-8_7.

Ishihara KL, Honda MDH, Pham DT, Borthakur D (2016) Transcriptome Analysis of Leucaena leucocephala and Identification of Highly Expressed Genes in Roots and Shoots. Transcriptomics 4:135. doi:10.4172/2329-8936.1000135

Dudley NS, Jones TC, James RL, Sniezko RA, Cannon P, and Borthakur D (2015) Applied disease screening and selection program for resistance to vascular wilt in Hawaiian Acacia koa, Southern Forests: a Journal of Forest Science, 77: 65-73, DOI: 10.2989/20702620.2015.1007263

Pal A, and Borthakur D (2015) Transgenic overexpression of Leucaena β-carbonic anhydrases in tobacco does not affect carbon assimilation and overall biomass.  Plant Biosystems. DOI:10.1080/11263504.2014.993739.

Ishihara K, Lee EW, Rushanaedy I, and Borthakur D (2015). Illumina-based de novo transcriptome analysis and identifications of genes involved in the monolignol biosynthesis pathway in Acacia koa. American Journal of Bioinformatics, 4(1): 7-27. doi: 10.3844/ajbsp.2015.7.27 Open Access.

Watanabe S, Borthakur D, Bressan A (2015) Localization of banana bunchy top virus and cellular compartments in gut and salivary gland tissues of the aphid vector Pentalonia nigronervosa.  Insect Science DOI: 10.1111/1744-7917.12211.

Negi VS, Bingham J-P, Li QX, Borthakur D (2014) A carbon-nitrogen lyase from Leucaena leucocephala catalyzes the first step of mimosine degradation. (Plant Physiology 164: 922-934 (Published online before print on December 2013, doi: http:/ / dx. doi. org/10. 1104/ pp. 113. 230870) Open Access.

Yafuso JT, Negi VS, Bingham J-P, Borthakur D (2014) An O-acetylserine (thiol) lyase from Leucaena leucocephala is a cysteine synthase but not a mimosine synthase. Applied Biochemistry and Biotechnology 173:1157–1168 DOI: 10.1007/s12010-014-0917-z

Pal A, Borthakur D (2014) Tissue-specific differential expression of two β-carbonic anhydrases in Leucaena leucocephala under abiotic stress conditions. J Appl  Biotechnol 2: 43-64

Negi VS, Bingham J-P, Li QX, Borthakur D (2013) midD-encoded ‘rhizomimosinase’ from Rhizobium sp. strain TAL1145 is a C–N lyase that catabolizes L-mimosine into 3-hydroxy-4-pyridone, pyruvate and ammonia. Amino Acids 44(6):1537-47. DOI 10.1007/s00726-013-1479-z.

Watanabe S, Borthakur D, Bressan A (2013) Lack of evidence for an interaction between Buchnera GroEL and Banana bunchy top virus (Nanoviridae). Virus Research 177: 198–102.  http://dx.doi.org/10.1016/j.virusres.2013.06.002

Adamski DJ, Dudley NS, Morden CW, Borthakur D (2013) Cross-amplification of non-native Acacia species in the Hawaiian Islands using microsatellite markers from Acacia koa.  Plant Biosystems 146: 24–32. DOI: 10.1080/11263504.2012.749958.

Pal A, Negi VS, Khanal S, Borthakur D (2012) Immunodetection of curcin in seed meal of Jatropha curcas using polyclonal antibody developed against curcin-L. Current Nutrition & Food Science 8: 213-219.

Rushanaedy I, Jones TC, Dudley NS, Liao RJF, Agbayani R, Borthakur D (2012) Chitinase is a potential molecular biomarker for detecting resistance to Fusarium oxysporum in Acacia koa.  Tropical Plant Biol. 5:244–252.  DOI 10.1007/s12042-012-9108-7.

Adamski DJ, Dudley NS, Morden CW, Borthakur D (2012) Genetic differentiation and diversity of Acacia koa populations in the Hawaiian Islands.  Plant Species Biology. 27: 181-190 (with cover page photo from our work) DOI: 10.1111/j.1442-1984.2011.00359.x

Pal A, Negi VS, Borthakur D (2012) Efficient in vitro regeneration of Leucaena leucocephala using immature zygotic embryos as explants. Agroforestry Systems 84:131–140  (DOI 10.1007/s10457-011-9438-8).

Singh VS, Pal A, Singh R, Borthakur D (2011) Identification of species-specific genes from Leucaena leucocephala using interspecies suppression subtractive hybridization.  Annals of applied Biology 159: 387–398  (doi:10.1111/j.1744-7348.2011.00506.x).

Walton CB, Jube S, Schrlemmer A, Patek PQ, Zimmerman DH, Rosenthal KS, Borthakur D (2010) Ex vivo stimulation assay for T-cell responses for tuberculosis using LEAPS-peptide heteroconjugates. Current Trends in Microbiology 6:1-12.

Jube SLR, Borthakur D (2010) Transgenic Leucaena leucocephala expressing the Rhizobium gene pydA encoding a meta-cleavage dioxygenase shows reduced mimosine content. Plant Physiol Biochem 48 (2010) 273-278

Jube S, Awaya J, Borthakur D (2009) Expression of Rhizobium pydA-pydB fusion gene in Nicotiana tabacum confers resistance to the toxic aromatic compound 3-hydroxy-4-pyridone. Biologia Plantarum 53 (2): 355-359, 2009

Jube S, Borthakur D (2009) Development of an Agrobacterium-mediated transformation protocol for the recalcitrant tree-legume Leucaena leucocephala using immature zygotic embryos. Plant Cell, Tissue and Organ Culture (PCTOC): Journal of Plant Biotechnology 96: 325-333.

Fredua-Agyeman R, Adamski D, Liao RJ, Morden C, Borthakur D (2008) Development and characterization of microsatellite markers for analysis of population differentiation in the tree legume Acacia koa (Fabaceae: Mimosoideae) in the Hawaiian Islands.  Genome 51: 1001-1015.

Walton CB, Inos ABH, Andres OA, Jube S, de Couet HG, Douglas JT, Patek PQ, Borthakur D (2008) Immunization with hybrid recombinant Mycobacterium tuberculosis H₃₇Rv proteins increases the T_H1 cytokine response in mice following a pulmonary instillation of irradiated mycobacteria.  Vaccine 26 26,4396-4402.

Kutin RK, Jenkins DM, Borthakur D (2008) Characterization of a Corynebacterium strain that can reduce nitrate from high strength nitrate medium. Bioremediation Journal. 12(3):168-172.

Awaya JD, Tittabutr P, Li QX, Borthakur D (2008) Pyruvate carboxylase is involved in metabolism of mimosine by Rhizobium sp. strain TAL1145. Archives of Microbiology190: 409-415.  DOI:10.1007/s00203-008-0384-4.

Tittabutr P, Awaya JD, Li QX, Borthakur D (2008) The cloned 1-aminocyclopropane-1-carboxylate (ACC) deaminase gene from Sinorhizobium sp. strain BL3 in Rhizobium sp. strain TAL1145 promotes nodulation and growth of Leucaena leucocephala. Systematic and Applied Microbiology 31:141-150.

Jube S, Borthakur D (2007) Expression of bacterial genes in transgenic tobacco: methods, applications and future prospects.  Electronic J Biotechnol. 10 (3): 452-467. DOI: 10.2225/vol10-issue3-fulltext-4     http://www.ejbiotechnology.info/content/vol10/issue3/full/4/4.pdf

Awaya JD, Walton C, Borthakur D (2007) The pydA-pydB gene produces an active dioxygenase-hydrolase that degrades 3-hydroxy-4-pyridone, an intermediate of mimosine metabolism.  Appl. Microbiol. Biotechnol 75(3):583-588.  DOI 10.1007/s00253-007-0858-3

Tittabutr P, Payakapong W, Teaumroong N, Boonkerd N, Singleton PW, Borthakur D (2006) The alternative sigma factor RpoH2 is required for salt tolerance in Sinorhizobium sp. strain BL3.  Res Microbiol.157: 811-818

Leary JK, Singleton PW, Scowcroft PG, Borthakur D (2006) Symbiotic diversity in the cosmopolitan genus Acacia.  Symbiosis 41 (3): 107-117

Payakapong W, Tittabutr P, Teaumroong N, Boonkerd N, Singleton PW, Borthakur D (2006) Identification of two clusters of genes involved in salt tolerance in Sinorhizobium sp. strain BL3. Symbiosis 41: 47-51

Tittabutr P, Payakapong W, Teaumroong N, Boonkerd N, Singleton PW, Borthakur D (2006) A histidine kinase sensor protein gene is necessary for induction of low pH tolerance in Sinorhizobium sp. strain BL3. Antonie Van Leeuwenhoek 89 (1): 125-134 (Online: December 8, 2005; DOI : 10.1007/s10482-005-9015-0)  

Leary JK, Hue NV, Singleton PW, D. Borthakur (2006) Soil acidification, nutrient depletion, and symbiotic nitrogen fixation are the major features of gorse (Ulex europaeus) infestation on volcanic soils in Hawaii. Biol. Fertility Soils. 42:215-223.  Published online: 28 June 2005

Awaya JD, Fox PM, Borthakur D (2005) pyd genes of Rhizobium sp. strain TAL1145 are required for degradation of 3-hydroxy-4-pyridone, an aromatic intermediate in mimosine metabolism.  J. Bacteriol. 187 (13): 4480-4487.

Past students

Ph.D.:

1. Kazue Ishihara (2016) Ph.D.  Mechanical stress induces resistance against wilt disease caused by Fusarium oxysporum in Acacia koa.
2. Archana Pal (2013) Ph.D. Functional characterization of chloroplastic and cytoplasmic β-carbonic anhydrase isoforms from Leucaena leucocephala
3. Vishal Negi (2007-2012) Ph.D.  Dissertation title:  Biochemistry of mimosine catabolism by enzymes from Rhizobium sp. TAL1145 and Leucaena leucocephala.
4. Sandro Jube (2005-2009) Ph.D. Dissertation subject: Genetic transformation of Leucaena leucocephala.
5. Rudolph Fredua-Agyeman (2005-2008) Ph.D. Dissertation subject: Genetic diversity in Acacia koa in Hawaiian Islands. 
6. Chad Walton (2002-06) PhD.  Dissertation subject: Recombinant hyrbrid vaccine development against Mycobacterium tuberculosis.
7. James Leary (2001-07) Ph.D. Dissertation subject: Bradyrhizobium inoculant of Acacia koa.
8. Jonathan Awaya (2001-05) Ph.D. Dissertation subject: Analysis of pyd gene cluster in Rhizobium.
9. Pakieli Kafusi (1999-05) Ph.D. dissertation: Regulation of exopolysaccharide synthesis in Rhizobium by an alternate sigma factor.
10. Paul Fox (1999-02) Ph.D. dissertation: Biochemistry and genetics of mimosine degradation in Rhizobium.          
11. Rongguan Jin (1995-1999) Ph.D. dissertation: Genetic engineering of cabbage and watercress with B.T. cry genes for insect resistance.
12. Muchdar Soedarjo (1993-1997) Ph.D. dissertation: Genetics of mimosine degradation by Rhizobium sp. strain TAL1145 that nodulates Leucaena.   
13. Nikhat Parveen (1991-1995) Ph.D. dissertation: Genetics of exopolysaccharide synthesis in Rhizobium sp. strain TAL1145 that nodulates tree legumes.

M.S. Plan-A (with thesis):

1. Jadd Correia (2012-14) M.S. Thesis title: Genetic transformation of Leucaena leucocephala.
2. Jannai Yafuso (2010-2013) M.S. Thesis title: Cysteine and mimosine biosynthesis catalyzed by cysteine/mimosine synthase from Leucaena leucocephala.
3. Dung Pham (2011-2013) M.S. Thesis title: Transcriptome analysis, biochemical characterization and tissue culture regeneration of Leucaena leucocephala.
4. Tyler Jones (2009-2011) M.S. Thesis title: Developing Fusarium wilt-resistant Acacia koa.
5. Daniel Adamski (2006-2008) M.S.  Thesis title:  Molecular phylogenetics and diversity of the Acacia koa complex based on DNA sequences and microsatellite markers.
6. Masaki Nasu (2003-2006) M.S.  Thesis subject: Developing molecular markers for detection of homozygous lyretail fin type in sword fish.
7. Sandro Jube (2002-2005) M.S. Thesis subject.  Expression of Rhizobium dioxygenase and hydrolase genes in tobacco.
8. Joy Anamizu (1999-2002) M.S. thesis:  Isolation, identification and characterization of mimosine-degrading soil bacteria isolated from the rhizosphere of Leucaena leucocephala.
9. Havila Saafi (2000-2001) M.S.  thesis: Tissue culture of Leucaena leucocephala.
10. Zerong You (1996-1998) M.S. Thesis: A stomatin-like protein from Rhizobium etli is required for nodulation competition on Phaseolus bean.
11. Jyothirmai Gubili (1995-1997) M.S. thesis: Isolation and characterization of uptake hydrogenase  genes from Anabaena sp. strain PCC7120.
12.  Xuefeng Gao (1992-1995) M.S. thesis:  Genetic analysis of nodulation competitiveness in Rhizobium  etli.
13.  Shahriar Pooyan (1990-1992): M.S thesis: Identification and characterization of chromosomal genes in Rhizobium  required for nitrogen fixation in beans.